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Cytosolic Ca2+-dependent Ca2+ release activity primarily determines the ER Ca2+ level in cells expressing the CPVT-linked mutant RYR2
Author(s) -
Nagomi Kurebayashi,
Takashi Murayama,
Ryosaku Ota,
Junji Suzuki,
Kazunori Kanemaru,
Takuya Kobayashi,
Seiko Ohno,
Minoru Horie,
Masamitsu Iino,
Fumiyoshi Yamashita,
Takashi Sakurai
Publication year - 2022
Publication title -
˜the œjournal of general physiology/˜the œjournal of general physiology
Language(s) - English
Resource type - Journals
eISSN - 1540-7748
pISSN - 0022-1295
DOI - 10.1085/jgp.202112869
Subject(s) - ryanodine receptor , ryanodine receptor 2 , catecholaminergic polymorphic ventricular tachycardia , cytosol , mutant , chemistry , calcium , hek 293 cells , biophysics , microbiology and biotechnology , biology , intracellular , receptor , biochemistry , enzyme , organic chemistry , gene
Type 2 ryanodine receptor (RYR2) is a cardiac Ca2+ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca2+ release, which tends to occur when [Ca2+]ER reaches a threshold. Mutations lower the threshold [Ca2+]ER by increasing luminal Ca2+ sensitivity or enhancing cytosolic [Ca2+] ([Ca2+]cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca2+]cyt-dependent activity of RYR2 and the threshold [Ca2+]ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca2+]cyt and [Ca2+]ER using fluorescent Ca2+ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER than WT cells. The [Ca2+]cyt-dependent activity at resting [Ca2+]cyt, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca2+]ER and Arest. In addition, lowering RYR2 expression increased the threshold [Ca2+]ER and a product of Arest, and the relative expression level for each mutant correlated with threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on the net Ca2+ release rate via RYR2. Modeling reproduced Ca2+ oscillations with [Ca2+]cyt and [Ca2+]ER changes in WT and CPVT cells. Interestingly, the [Ca2+]cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced [Ca2+]cyt-dependent activity without requiring modulation of the [Ca2+]ER sensitivity of RYR2.

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